1. Field of the Invention
The present invention relates to an apparatus and method for measuring acidity of free fatty acids contained in oil such as edible oil; citric acid, malic acid or tartaric acid contained in fruit juice or fruit juice drinks; organic acids contained in alcoholic drinks; organic acids such as chlorogenic acids contained in coffee; or free fatty acids released from substrate oil by enzyme reaction of esterase in serum.
2. Description of the Related Art
Recently, it has been demanded that food have a quality above certain criteria for health and safety reasons. Among various components contained in food, acid has a significant influence on the quality of the food. Moreover, food having a relatively low acid content has been preferred recently. Acidity of various types of food has a considerable influence on the quality of food. The degree of influence and the method for measurement of the acidity vary in accordance with different types of food. Hereinafter, conventional methods for measuring the acidity of (1) edible oil, (2) fruit drinks such as juice, (3) alcoholic drinks such as whisky, "sake" or wine, (4) coffee, and (5) juice contained in fruits such as oranges or grapes will be described.
(1) Edible oil:
Diet in Japan is rapidly changing. A first change is the inclination for instant food. A second change is the diversification of tastes, which is represented by the increase in sales of processed food. Specifically, the increased preference for precooked food can be considered as reflecting the times, and a greater variety of processed food are being consumed in greater quantities. Especially, consumption of fried food has shown a remarkable increase. Some of the reasons for this increase are that fried food is preferred in terms of taste and is relatively less perishable. Even in the case of fried food, though, after being left in conditions where the influence of the temperature or light is significant, fats and oils are spontaneously oxidized by oxygen contained in the air, resulting in generation of putrid smell and deterioration of quality. For these reasons, general concern has grown regarding putrefaction and deterioration of edible fats and oils and processed food cooked with oils. For example, a regional food authentification system for fried bean curd has been started or a restriction on fried snacks has been introduced. Legal restriction of deterioration of fats and oils has been discussed in terms of establishing guidelines for boxed lunches and ready-made dishes sold in stores.
In order to examine the degree of deterioration of such fats and oils, especially heated fats and oils, there are several methods available for analysis, e.g., measurement of the acid value, the peroxide value, viscosity, and iodine value. Considering that food deterioration is mostly influenced by the temperature, humidity and light and that the acidity significantly changes during the initial stage of deterioration, measurement of acid value by which the acidity is directly measured, is appropriate and commonly used.
(2) Fruit drinks such as juice:
Fruit juice drinks are obtained by subjecting the fruits to squeezing. More fruit juice drink products are produced from concentrated fruit juice or frozen fruit juice rather than from fresh squeezed fruit juice.
For example, orange juice products are produced by removing blighted and unripe oranges, then washing the peel, pressurizing the oranges to take out the pulp and juice, and removing the pericarp, and the like from the juice. At this point, the sugar concentration, the acidity and the like are adjusted in conformity with the Japanese Agricultural Standards. At this point, the acidity is measured. In the case of producing orange juice from concentrated juice or frozen juice by adding water, the acidity is also measured at the time of adding water.
(3) Alcoholic drinks:
There are various types of alcoholic drinks produced in various manners. For example, distilled liquor represented by whisky or "shochu" is produced by repeating distillation to increase the yield of ethanol. Drinks represented by "sake" or wine are produced by fermentation and filtering. There are still other types of alcoholic drinks, e.g., effervescent liquor such as fruit wine or beer. Either type of alcoholic drink is subjected to acidity measurement during a production process in order to assure the quality.
(4) Coffee:
There are many types of substances as mentioned below providing a sour taste which mainly determines the taste of coffee. The acid content is important as a criterion for evaluating the sour taste of coffee. A representative type of acid contained in coffee is chlorogenic acids. The acid content thereof changes even while the coffee beans are roasted. Other substances such as coffee acid, quinic acid, and citric acid which are associated with the sour taste of coffee. Although the amount of each acid is very small, the delicate balance of the acid combination and the total amount of acid are considered to determine the sour taste in coffee.
(5) Juice contained in fruits such as oranges:
During the cultivation of oranges, especially during cultivation in a greenhouse, drying the inside of the oranges by withholding water is performed in order to increase the sugar level. According to this method, the concentration of sugar and acid in the fruit juice is increased by restricting the amount of water. However, as the sugar level increases in the fruit juice, the pleasant taste is enhanced; whereas as the acidity increases, the taste is worsened. Accordingly, after the inside of the oranges is dried by withholding water, the acid is consumed by causing the oranges to respire using an appropriate amount for water and an appropriate temperature while monitoring the acidity.
As described above, acidity is measured during the production of various types of food. There are a variety of methods available. Conventional acidity measuring methods are defined by, for example, the standard methods for analysis of oils and fats in the Official and Tentative Methods of the American Oil Chemists' Society, the Standard Methods for the Analysis of Oils, Fats and Derivatives of the Japan Oil Chemists' Society, the Japanese Agricultural Standards, the Japanese Industrial Standards, the Test for Fats and Fixed Oils of the Japanese Pharmacopoeia, the Standard Methods of Analysis for Hygienic Chemists, and the Potable Water Test Method. All of these methods are based on a neutralization titration method using phenolphthalein as an indicator. The neutralization titration methods defined in the Potable Water Test Method and the Standard Methods for the Analysis of Oils, Fats and Derivatives of the Japan Oil Chemists' Society will be described.
In the Potable Water Test Method, the acidity is defined as the amount in milligrams of calcium carbonate contained in 1 liter of sample. In practice, the acidity is obtained in the following manner.
One hundred milliliters of sample water is taken. Next, about 0.2 mL of phenolphthalein indicator solution is added to the test water, and then a solution of 0.02 mM sodium hydroxide is added thereto. The container containing the resultant mixture is sealed and lightly shaken. After the pink color disappears, another solution of 0.02 mM sodium hydroxide is added, and the container is sealed and lightly shaken. The titration is continued until the faint but permanent pink color is visually observed even after shaking, and this point is defined as the end point of the neutralization. The volume a of sodium hydroxide in milliliters at the end point is obtained. The acidity is calculated by the formula: EQU Acidity (mg/L of calcium carbonate)=a.times.10
The acidity of the tap water is indicated by the amount of calcium carbonate in milligrams per liter as described above. The acidity of other representative acid-containing substances are indicated as follows.
The acidity of oranges is converted and indicated by the amount in weight percent of citric acid. The acidity of grapes is indicated by the amount in weight percent of tartaric acid. The acidity in fats and oils is, as described in detail below, indicated by an acid value, which is the amount in milligrams of potassium hydroxide required to neutralize the free fatty acid contained in 1 gram of fats and oils. As described above, the indicator representing the acidity is defined for each different type of substances.
The neutralization titration method defined in the Standard Methods for the Analysis of Oils, Fats and Derivatives will be described in the case where it is used to determine the acidity of fats and oils. In the Standard Methods for the Analysis of Oils, Fats and Derivatives, the acid value is defined as the amount in milligrams of potassium hydroxide required to neutralize the free fatty acid contained in 1 gram of fats and oils.
The acidity of a liquid sample is measured in the following manner. An amount of the sample is taken in accordance with an estimated acid value (for example, 20 grams for the estimated acid value of 1 or less, 10 grams for the estimated acid value of more than 1 but equal to or less than 4, and 2.5 grams for the estimated acid value of more than 4 but equal to or less than 15). The accurate intended amount of sample is measured and put into an Erlenmeyer flask. One hundred milliliters of neutral solvent is added, and shaken until the sample is completely dissolved. The neutral solvent herein is obtained by adding about 0.3 mL of phenolphthalein indicator solution to a 1:1 mixed solvent of ethylether and ethanol, and neutralizing the resultant substance by 1/10 N potassium hydroxide-ethanol solution immediately before use.
The acidity of a solid sample is measured in the following manner. The sample is melted by heat in a water bath. Then, a solvent is added and the sample is dissolved. The resultant substance is titrated by 1/10 N potassium hydroxide-ethanol standard solution, and the time when the pink color of the indicator continues for 30 seconds is defined as the end point of neutralization. The amount in milligrams of potassium hydroxide is obtained by calculation.
The acid value of the fats and oils such as edible oil can be obtained through the measurement of the fatty acid by voltammetry rather than the neutralization titration. According to this method, which is disclosed in Japanese Laid-Open Publication No. 5-264503, the amount of the fatty acid in the electrolyte solution containing both free fatty acid and a naphthoquinone derivative is measured by voltammetry at a controlled potential. This method utilizes the property that the current value of the pre-peak of voltammetric reduction of the naphthoquinone derivative changes in proportion to the concentration of all types of free fatty acid, including lower fatty acid such as formic acid and higher fatty acid such as oleic acid and linoleic acid, and that the value obtained by overlapping the current values of different types of fatty acids corresponds to the total concentration of the fatty acids. In other words, the acid concentration is measured by measuring the current value of the pre-peak of voltammetric reduction of the naphthoquinone derivative. FIG. 17 is a graph illustrating the current vs. potential relationship for acidity measurement by voltammetry of an electrolyte solution containing a naphthoquinone derivative. The solid line in FIG. 17 indicates the data obtained by such a method of measurement. In FIG. 17, the axis of abscissa represents the potential of a working electrode with respect to the potential of a reference electrode in the case where the reference electrode is formed of silver--silver chloride and the working electrode is formed of .phi.3 glassy carbon. The axis of ordinates represents the value of the current flowing in the circuit. It should be noted that the current value varies in accordance with various conditions such as the surface area of the working electrode while the current value slightly fluctuates in accordance with the acid concentration; the fluctuation is negligible. In FIG. 17, letter A indicates the pre-peak depending on the acid concentration, and letter C indicates the main peak of the naphthoquinone derivative.
In order to measure the acid value of fats and oils by the method disclosed in Japanese Laid-Open Publication No. 5-264503, nitrogen gas or the like needs to be supplied to the electrolyte solution so as to remove the oxygen dissolved in the electrolyte solution. The data represented by the solid line in FIG. 17 is obtained in the state where the dissolved oxygen is removed. Unless the dissolved oxygen is removed, the current for reducing the dissolved oxygen flows and thus it becomes difficult to determine the current value of the pre-peak of reduction. The dotted line in FIG. 17 represents the reduction curve in the case where the dissolved oxygen is not removed. As can be seen in FIG. 17, the reduction curve of the oxygen and the pre-reduction curve overlap and thus the pre-peak of the fatty acid can hardly be determined. The reasons will now be described.
FIG. 18 shows a pre-peak curve of acidity measurement by voltammetry of a conventional electrolyte solution containing a naphthoquinone derivative. FIG. 19 shows a peak curve of acidity measurement by voltammetry of the conventional electrolyte solution containing a naphthoquinone derivative. FIG. 20 shows an oxygen reduction curve obtained in acidity measurement by voltammetry of an electrolyte solution. When the voltammetry is performed after removal of the dissolved oxygen in the electrolyte solution, the potential-current curve (hereinafter, referred to as the "voltammogram") which represents the result of synthesizing the pre-peak curve and the peak curve can be obtained, and thus the pre-peak appears clearly. In contrast, when the dissolved oxygen is not removed, the voltammogram represents the result of synthesizing the pre-peak curve, the peak curve and the oxygen reduction curve. This is a conceivable reason why the pre-peak does not appear clearly. The acidity measurement is difficult by the conventional method unless the electrolyte solution is deoxidized. In order to deoxidize the electrolyte solution, the acidity measuring apparatus is provided with a device for continuously supplying gas (e.g., nitrogen) to the acidity measuring apparatus. Such a structure enlarges the size of the acidity measuring apparatus, which prevents this method from being put into practical use.
In the field of measuring the lipid component in serum, a different substance from the substance to be measured is measured after several stages of reaction, since there is conventionally no appropriate method available for directly measuring the fatty acid or organic acid in the solution. The measurement of serum will be described in detail below.
The number of people obtaining a value outside the normal value range during the examination of the concentration of cholesterol, neutral fat (glycerin fatty acid ester), or phospholipid in serum is rapidly increasing due to increased consumption of American and European style food, increasing opportunities of drinking alcoholic drinks, lack of physical exercise, stress, or the like. Among the lipid components, the cholesterol level is used as a risk factor indicator of lifestyle-related diseases such as diabetes, arterial sclerosis, or hypothyroidism. The value of the neutral fat (glycerin fatty acid ester) is used as a risk factor indicator of lipid dysbolism, cerebrovascular accident, cardiac infarction, angina pectoris, and diabetes. The value of the lipid components is also used as a risk factor indicator of lipid dysbolism, cerebrovascular accident, cardiac infarction, angina pectoris, and diabetes, and also acts as a health risk factor indicator of hepatopathy obliterans, hyperthyroidism, and fulminant hepatitis.
Conventionally, the lipid components used as an indicator of the above-mentioned diseases are measured mainly by an enzyme method. In other words, a lipid component is decomposed into fatty acid and other components by an enzyme, and the components which are not fatty acids are measured. For example, the neutral fat is measured in the following manner. Serum is treated by lipoprotein lipase as an enzyme to decompose the neutral fat into glycerol and trifatty acid. Then, glycerolkinase as an enzyme for treating the glycerol, magnesium ions and adenosine triphosphatase (ATP) are added to decompose the glycerol into glycerol-1-phosphate and adenosine diphosphatase (ADP). Next, glycerol-1-phosphate oxidase as an enzyme for treating the glycerol-1-phosphate is added to decompose the glycerol-1-phosphate into dihydroxyacetone-1-phosphate and hydrogen peroxide. Finally, the peroxidase for treating the hydrogen peroxide and 4-aminoantipyrine and dimethyl aniline are added to generate a red quinone dye. The amount of the red quinone dye (corresponding to the amount of hydrogen peroxide) is measured, and the amount of neutral fat is obtained by calculation. The results of the above-described reactions are obtained with about 3 to 20 .mu.L of serum.
The reason why the hydrogen peroxide obtained after the plurality of reaction stages is measured is that the neutralization titration and the technology disclosed in Japanese Laid-Open Publication No. 5-264503 are both difficult to apply to serum treated by an enzyme such as lipoprotein lipase, since the change of color caused by the indicator is difficult to read in the case of serum, and also since the serum contains oxygen.
As described above, by the conventional acidity measuring method, which uses the neutralization titration method, the observer monitoring the color change caused by the phenolphthalein indicator determines when the end point is reached. Accordingly, the end points vary and thus the acidity varies depending on the operator.
According to the neutralization titration defined by the Standard Methods for the Analysis of Oils, Fats and Derivatives used for measuring the fatty acid, a mixed solution containing ether and ethanol is used as the neutral solvent. Ether having a boiling point of as low as 34.6.degree. C. is difficult to handle. Moreover, in the case where the sample has a dark color, such as oil which has been used for frying a large amount of food, or juice or wine which are originally dark colored, the color change caused by the phenolphthalein indicator near the end point cannot be accurately recognized. Thus, the measured value fluctuates. Furthermore, one cycle of measurement requires as much as 100 mL of neutral solvent and as much as 10 grams of sample. Also, the more measurement is performed, the greater the cost.
According to the above-described technology for measuring the lipid component in serum, treatment by enzyme is performed in 3 or 4 steps for any lipid component. Different components require different enzymes and a large amount of samples, which makes the operation excessively troublesome. Moreover, the results cannot be obtained until 3 to 4 stages of reactions are finished. In the case where an error occurs, it takes time to find in which stage the error occurred. Hydrogen peroxide as a target of measurement is unstable, and thus an error can possibly be generated unless the operation is performed quickly.
The technology disclosed in Japanese Laid-Open Publication No. 5-264503 has the following problem. In the case where a naphthoquinone derivative is used without removing oxygen from the electrolyte solution in order to reduce the size of the acidity measuring apparatus, a current is generated by the reduction of the oxygen dissolved in the solution, and the value of such a current overlaps the current value of acid to be measured as illustrated in FIG. 17. In the case where a small amount of acid is measured, the measured value fluctuates depending on the amount of dissolved oxygen, thus decreasing reliability. When a device for supplying gas is provided for removing the oxygen, the entire apparatus is enlarged and becomes difficult to handle. Thus, such an apparatus is difficult to put into practical use.
Moreover, by the above-described technology disclosed in Japanese Laid-Open Publication No. 5-264503, the electrolyte solution contains a protic organic solvent. The oil actually used and deteriorated by heat is hardly dissolved in the protic organic solvent (e.g., propanol, methanol, or ethanol). Even the use of a stirrer does not work. Accordingly, after the solution containing oil is stirred sufficiently, the oil layer is separated and removed by centrifugation, and the remaining solution is used as the electrolyte solution. The use of a centrifuge inevitably enlarges the entire apparatus. Furthermore, since the electrolyte solution is extracted from the solution containing oil, the measured value fluctuates when the solution containing oil is not stirred sufficiently. Accordingly, a reliable acidity measuring apparatus cannot be realized as in the case with the neutralization titration method. The technology involves difficult problems to solve before it can be embodied as an actual measuring apparatus although being superior in terms of principle.
As disclosed in Japanese Laid-Open Publication No. 5-264503, quinones other than naphthoquinone are conventionally considered to be too unstable to be used for accurate acidity measuring. The quinones are unstable to light even in the form of crystals, and are especially susceptible to photolysis when they are in the form of a solution. Specifically, the color of a solution containing benzoquinone changes into reddish purple when exposed to sunshine, and new absorption maximums are generated in the range of the ultraviolet and visible light. Since such a decomposition is facilitated in the case of an organic solvent, use of benzoquinone is conventionally considered to cause photolysis to prevent accurate measurement. Accordingly, the method of using a naphthoquinone derivative is conventionally considered to be the only possible method, and the problems of the dissolved oxygen involved in the use of the naphthoquinone derivative are difficult to solve.